1. Field of the Invention
The present invention relates to a coating composition for culturing cells, which enhances the adhesive property and growth of adhesive cells. The present invention also relates to a serum-free culturing method of animal adhesive cells.
2. Prior Art
In the field of biotechnology, techniques of cell culture have been developed extensively for the production of such substances that are difficult to obtain by synthesis as antibodies, hormones, enzymes, nucleic acids and other physiologically active compounds by utilizing animal cells. These animal cells are divided into suspended cells and adhesive cells, and the former are relatively easy to culture since they can proliferate in suspension of the medium. By contrast, the latter requires to adhere on the inner surface of culture vessel and can grow only on the solid surface. Therefore, the state and characteristics of the inner surface of the vessel or carrier onto which the cells adhere influence greatly on the cell growth and functions of the cells.
Previously, glass or plastic vessel for cell culture have been used, but their intact surface lacks sufficient hydrophilicity so that cell attachment onto the surface and cell growth has not been satisfactory.
For overcoming the difficulty, a method of making the inner surface of a cell culture vessel hydrophilic by special plasma treatment (see Japanese Patent Laid-Open Publication No. 76570/1990), and another one in which the inner surface is coated with cell adhesive proteins such as collagen, gelatin and fibronectin (see Japanese Patent Laid-Open Publication No. 71884/1983), have been commonly practiced.
However, the plasma treatment requires expensive plasma generating and gas exchanging apparatus and a long time with the result of high operation cost. In addition, since the treatment is done by batches, there may occur difference in some lots. The plasma-treated vessels don't have sufficient thermostability for heating treatment such as sterilization in autoclave. With some kind of cells, their adhesive and proliferative properties are rather deteriorated (see Japanese Patent Laid-Open Publication No. 109769/1988).
Further, purification of adhesive proteins such as collagen and gelatin to a high quality grade for use in cell culture requires complicated procedures, special apparatus and expensive reagents with the result of raising the cost of the final product. In addition, the coated vessels are short in thermostability for sterilization in autoclave or other heating treatments, and sterilization by .gamma.-rays causes partial degradation of the proteins to deteriorate the cell adhesion (see Japanese Patent Laid-Open Publication No. 234670/1990).
In addition, purification of collagen to a degree for use in cell culture requires considerable steps of processing and still a satisfactory purity cannot be achieved usually. Impurities in collagen, often are causes of contamination in the physiologically active substances produced by the cells. Further, many enzymes produced by cells by themselves not only make a cause of contamination but also work in long culture to decompose collagen causing release of the adhered cells from inner surface of the culture vessel. Further more, there are such problems as considerable difference in impurities among lots, and low thermostability of collagen makes impossible to sterilize the coated vessels in autoclave (see Japanese Patent Laid-Open Publication No. 291260/1990).
On the other hand, conventional methods for coating the inner surface of cell culture vessel with a synthetic polymer are, for example, the method in which a synthetic polymer is molded into a film which can be used as it is for cell culture, and that a synthetic polymer is suspended or dissolved in benzene, dioxane, N,N'-dimethylformamide or other organic solvent, the suspension or solution is applied over the inner surface of cell culture vessel, then the solvent is removed and the vessel is sterilized by autoclaving or .gamma.-rays irradiation (see Japanese Patent Laid-Open Publication No. 89179/1983). However, in the former method, a new apparatus is required and handling is time-consuming, while in the latter method, required to allow the vessel to stand for a definite time for coating of the polymer over the surface, and then the solvent is required to be removed by suction or drying with heating before the vessel is sterilized. When a water-soluble polymer is used as a coating polymer, there may occur that releasing and dissolving of the polymer into the medium during the cell culture. When a synthetic polymer dissolved in such an organic solvent as benzene, dioxane and N,N'-dimethylformamide is used for coating over the surface of culture vessel or other materials, ordinary commercially available culture vessels made of polystyrene or other plastic materials may be dissolved by the organic solvent and so the culture vessels to be used are limited to those made of organic solvent-resistant materials.
It is also noted that due to the fact that animal adhesive cells grow only when they adhere onto inner surface of culture vessel, culture of the cells is usually done by employing a serum-containing medium. Serum is comprising a variety of components including vitronectin that works to make the cells to be able to adhere onto the surface of the culture vessel.
It must be pointed out, however, serum is very expensive composition, the components are varying in lots and volume of one lot is limited. Therefore, problems are also present that complicated procedures are required at every change of the lot for check of quality and regulation and management of the culture conditions according to the quality. In addition, since serum is a mixture containing numerous substances with physiological activities which are released from blood cells and vascular endothelial cells, analysis or utilization of the products of cultured cells in a serum-containing medium requires procedures for high purification.
To solve the above discussed problems establishment of a serum-free culturing method of animal adhesive cells is desired.
Previously a number of serum-free culturing method of animal adhesive cells have been proposed. Reported examples are a method of culturing vascular endothelial cells in a serum-free medium to which heparin and a cell growth factor are added (see Japanese Patent Laid-Open Publication No. 187083/1989), a method of culturing vascular endothelial cells in a serum-free medium to which zinc or copper salt is added (see Japanese Patent Laid-Open Publication No. 97379/1990) and a method of a serum-free culturing method of fibroblasts by using a specific polystyrene-type polymer as a substrate (see Japanese Patent Laid-Open Publication No. 279787/1988). However, each of these methods has a limitation in cell species and so lacks universal applicability.